Oligonucleotides and kits for detection of htlvi and htlvii viruses by hybridization

ABSTRACT

The presence or absence of a nucleic acid sequence of an isolate of HTLVI and/or HTLVII in a sample containing one or more nucleic acids and suspected of containing such sequence can be detected by amplifying the sequence using primers to form extension products as templates and detecting the amplified product if it is present. This may be accomplished by adding a labeled hydridization probe to the amplified product, either free in solution or after immobilization on a solid support.

BACKGROUND OF THE INVENTION

The present invention relates to a process for detecting the presence orabsence of a conserved, nucleotide sequence of a virus related to humanT cell leukemia virus-types I and II (HTLVI and II). This invention alsorelates to a kit for such detection having primers and a labeledhybridization probe.

A family of T cell tropic retroviruses, known as human T cell leukemiaviruses (HTLV), is known to be involved in the pathogenesis of certain Tcell neoplasms. Currently, there exist three known types of HTLV. Thefirst, type I (HTLVI), is an oncovirus that has been linked to a humanadult T-cell leukemia-lymphoma (ATLL) that is found in Japan, theCaribbean region, and Africa. The second, type II (HTLVII), is anoncovirus that has been isolated from two patients having a T-cellvariant of hairy cell leukemia. See M. Popovic et al., Science,224:497-500 (1984) and Rosenblatt, J. D. et al., New Eng. J. Of Med.,August, 1986. The third, type III (HTLVIII), is a lentivirus and is theaetiologic agent responsible for acquired immune deficiency syndrome(AIDS), a transmissible disorder of the cellular immune system resultingin frequently fatal opportunistic infections.

The current immunodiagnostic tests to identify sera with antibodies tothe HTLV-associated virus(es) such as AIDS (see U.S. Pat. No. 4,520,113to Gallo et al.) are being used in blood banks to eliminate potentiallyinfectious blood. See also WO 86/01834 published Mar. 27, 1986(University of California) for retroviral polypeptides useful inpreparing monoclonal antibodies to detect retroviruses in the HTLVfamily Because the viruses may reside as a DNA copy without producingsignificant quantities of viral particles, a direct immunologicalapproach to detect HTLVI and II-related viruses may prove unsuccessfulin a significant fraction of persistently infected asymptomaticindividuals. Because the number of virus particles in the infectedtissues and blood may be few (due to viral quiescence), direct detectionof viral particles or RNA/DNA may be difficult, if not impossible,without co-culturing the infected cells with a permissive T cell line.

U.S. Pat. No. 4,683,202 describes a process for amplifying nucleic acidsequences to facilitate detection thereof, as by using a labeled RNA orDNA hybridization probe. In this process primers are used to obtainprimer extension products which are used as templates to synthesizeadditional complementary strands in the presence of nucleotides. Theabove-mentioned patent application also describes a technique wherebyafter a probe is hybridized to the desired sequence, a restrictionenzyme is added to cleave the hybrid at a site within the desiredsequence, and the restriction digest is then analyzed for labeledfragments. U.S. Pat. No. 4,683,194 and Saiki et al., Biotechnology,3:1008-1012 (1985) describe this latter technique in greater detail.Both patent applications illustrate use of the process for detectinggenetic diseases such as sickle cell anemia and β-thalassemia. Thesemethods and the process for amplifying nucleic acid sequences are alsodisclosed in Saiki et al., Science, 230, 1350-1354 (1985), thedisclosure of which is incorporated herein by reference.

A review article by Landry et al., Clin. Lab. Med. (1985) 5, 513-529describes the field of nucleic acid hybridization as applied to virusdetection. W086/01535 published Mar. 13, 1986 and EP 173,529 publishedMar. 5, 1986 disclose molecular cloning of HTLVIII and use of the cloneas a probe to detect AIDS. Further, EP patent publication 173,339,published Mar. 5, 1986, discloses a genetic analysis using a DNA probeto detect infections by foreign microbes. EP 185,444, published June 25,1986, discloses a recombinant peptide for use as a probe to detect theHTLVIII virus in cell lysates. Oncor Inc. announced in September, 1986that it has developed a radioactive blood test to detect the AIDS virus.Finally, copending U.S. application Ser. No. 935,581, filed Nov. 26,1986, now abandoned, which is a continuation-in-part of Ser. No. 818,127filed Jan. 10, 1986, now abandoned disclosed a method for detecting AIDSviruses using the amplification procedure described above together witha hybridization probe. This application is related to U.S. Ser. No.934,955, now abandoned, file Nov. 26, 1986, entitled "Detection ofViruses by Amplification and Hybridization."

Use of a hybridization probe to detect the oncoviruses HTLVI and II mayallow identification of those individuals who are persistently infectedbut are not producing virus or individuals who are antibody negative butculture positive, and to detect infected cells without the need toculture the virus. Increasing the viral nucleic acid copy number of thevirus by amplification will facilitate the identification of viralnucleic aicd in infected individuals.

SUMMARY OF THE INVENTION

The present invention involves a process for detecting or monitoring forthe presence or absence of a nucleic acid sequence which issubstantially conserved among the isolates of HTLVI or HTLVII nucleicacids or both HTLVI and HTLVII nucleic acids and specific to the nucleicacids in HTLVI or HTLVII or both HTLVI and HTLVII isolates and whichnucleic acid sequence is suspected of being contained in a sample, whichprocess comprises:

(a) treating the sample, together or separately, with an oligonucleotideprimer for each strand of the nucleic acid sequence, four differentnucleoside triphosphates, and an agent for polymerization, underhybridizing conditions, such that for each strand of the nucleic acidsequence an extension product of each primer is synthesized which iscomplementary to each nucleic acid strand, wherein said primer(s) aresubstantially complementary to each strand of the nucleic acid sequencebeing detected or monitored, such that the extension product synthesizedfrom one primer, when it is separated from its complement, can serve asa template for synthesis of the extension product of the other primer;

(b) treating the sample under denaturing conditions;

(c) treating the product of step (b) with oligonucleotide primers suchthat a primer extension product is synthesized using each of the singlestrands produced in step (b) as a template, resulting in amplificationof the specific nucleic acid sequence or sequences if present; and

(d) determining if the sequence to be detected is present in the sample.

One way to detect the product is by adding to the product of step (c) alabeled probe capable of hybridizing with the amplified nucleic acidsequence; and determining whether the probe has hybridized to anamplified sequence in the nucleic acid sample. In one embodiment, thisdetermination can be made by:

(1) digesting the hybridized mixture with a restriction enzymerecognizing a site within the sequences in the probe; and

(2) detecting whether the restriction digest contains a restrictionfragment correlated with the presence of the HTLVI or HTLVII sequence.

Before step (a) the nucleic acids in a patient sample may be extractedtherefrom so that the sample being treated is actually the mixture ofthe extracted nucleic acids. In addition, the sample being treated instep (a) need not be subjected beforehand to a process wherein the virusin the sample is cultured.

In another embodiment, the invention herein relates to a kit fordetecting or monitoring for the presence or absence of a nucleic acidsequence which is substantially conserved among the isolates of HTLVI orHTLVII nucleic acids or both HTLVI and HTLV II nucleic acids andspecific to the nucleic acids in HTLVI or HTLVII or both HTLVI andHTLVII isolates and which nucleic acid sequence is suspected of beingcontained in a sample, which kit comprises:

(a) one oligonucleotide primer for each strand of the nucleic acidsequence to be detected, which primer or primers are substantiallycomplementary to each strand of each specific nucleic acid sequence suchthat an extension product synthesized from one primer, when it isseparated from its complement, can serve as a template for the synthesisof the extension product of the other primer; and

(b) a labeled probe capable of hybridizing with the nucleic acidsequence.

Preferably, the kit also contains an agent for polymerization, fourdifferent nucleosides, and a means for detecting hybrids of the probeand sequence.

The test kit herein may be used in research tests, clinical tests andother diagnostic applications. In addition, it can be used to detectinfected cells without culturing the virus, a feature useful inmonitoring patients treated with various therapeutic agents to resolvethe infection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a process and kit for detecting ormonitoring for a nucleic acid sequence associated with either or both ofthe HTLVI and HTLVII viruses in a sample of nucleic acid(s) suspected ofcontaining the sequence. Isolates of the HTLVI and HTLVII viruses havebeen sequenced. The sequence to be amplified must be specific to theHTLVI and/or II virus, i.e., not react with HTLVIII or other non-HTLVIor II viruses.

The entire genome of the HTLVI virus is provided by Seiki et al., Proc.Natl. Acad. Sci. USA 80:3618-3622 (1983), the disclosure of which isincorporated herein by reference. The entire genome of the HTLVII virusis provided by Shimotohno et al., Proc. Natl. Acad. Sci. USA82:3101-3105 (1985), the disclosure of which is incorporated herein byreference.

The term "substantially conserved" as applied to the sequence to bedetected signifies that the sequence must be sufficiently complementaryto the nuclei acids in the virus being detected to initiatepolymerization at least at room temperature in the presence of an agentfor polymerization and the four nucleoside triphosphates.

The primers used will be oligonucleotides of any length and sequence soas to provide specific initiation of polymerization on a significantnumber of nuclei acids in the HTVLI and/or II viruses. Specifically, theterm "primer" as used herein refers to a molecule comprised of two ormore deoxyribonucleotides or ribonucleotides, preferably more thanthree, which is capable of acting as a point of initiation of synthesiswhen placed under conditions in which synthesis of a primer extensionproduct which is substantially complementary to a nucleic acid strand isinduced, i.e., in the presence of nucleoside triphosphates and an agentfor polymerization such as DNA polymerase and at a suitable temperatureand pH. The primer is preferably single stranded for maximum efficiencyin amplification, but may alternatively to double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent for polymerization. The exact lengths of the primers will dependon many factors, including temperature, buffer, nucleotide compositionand source of primer. For purposes herein, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be "substantially" complementary toeach strand of the specific sequence to be amplified. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands under conditions which allow the agent forpolymerization to perform, i.e., the primers have sufficientcomplementarity with the sequence of the strand to be amplified tohybridize therewith and thereby form a template for synthesis of theextension product of the other primer. The primers may contain somemismatches with the strand.

One may select the sequence being amplified from among the region thatis substantially conserved among the HTLVI and HTLVII viruses.Therefore, the primers and probes may be identified and selected by anysuitable means. This may be done manually by comparing the regions ofthe published nucleic acid sequences of the HTLVI and HTLVII viralgenomes. The nucleotide sequence homologies between the X regions of theHTLVI and HTLVII viruses have been published by Shimotohno et al., Proc.Natl. Acad. Sci. USA 81:6657-6661 (1984), the entire disclosure of whichis incorporated herein by reference. Another method is to use a computerprogram to compare the sequences. For this purpose, a commercial programwith the underlying computer algorithm supplied by National BiomedicalResearch Foundation using a dot matrix may be conveniently employed.This program involves inputting the nucleic acid sequences of the HTLVIand II viruses and defining a window size for base pair homology. Theprogram employs graphics to compare the sequences on different axes, anda dot appears where there is at least substantial homology. Preferably,the window size is greater than six bases.

The X region of the genome is most conserved among the coding regions inthe two viruses. Because this is most conserved among the codingregions, it is the preferred region from which to select primers andprobes for detecting the sequence. Regions of the viral genome that donot encode proteins can also be used to determine a sequence for theprimers to be used. For purposes herein, to maximize sensitivity andspecificity, the sequence being detected is homologous with a sequenceof a length sufficient to allow specific priming which is substantiallyconserved among the related viruses, particularly at the restrictioncleavage site if a probe and restriction enzyme are employed.

The techniques used for amplifying and thereafter detecting the productare described in detail in U.S. Pat. Nos. 4,683,202 and 4,683,194identified above, Saiki et al., Biotechnology, supra and Saiki et al.,Science, supra, the entire disclosures of which are incorporated hereinby reference. In general, the amplification process involves anenzymatic chain reaction for preparing, in exponential quantitiesrelative to the number of reaction steps involved, a specific nucleicacid sequence, given that the ends of the required sequence are known insufficient detail that oligonucleotide primers can be synthesized whichwill hybridize to them, and that a small amount of the sequence isavailable to initiate the chain reaction. One primer is complementary tothe negative (-) strand and the other is complementary to the positive(+) strand. Annealing the primers to denatured nucleic acid followed byextension with an enzyme such as the large fragment of DNA Polymerase I(Klenow) and nucleotides results in newly synthesized + and - strandscontaining the target sequence. Because these newly synthesizedsequences are also templates for the primers, repeated cycles ofdenaturing,, primer annealing and extension results in exponentialaccumulation of the region defined by the primer. The product of thechain reaction will be a discrete nucleic acid duplex with terminicorresponding to the ends of the specific primers employed.

The amplification process is illustrated diagrammatically below, wheredouble-stranded DNA containing the desired sequence [S] comprised ofcomplementary strands [S⁺ ] and [S⁻ ] is utilized as the nucleic acid.During the first and each subsequent reaction cycle extension of eacholigonucleotide primer on the original template will produce one newssDNA molecule product of indefinite length which terminates with onlyone of the primers. These products, hereafter referred to as "longproducts," will accumulate in a linear fashion; that is, the amountpresent after any number of cycles will be proportional to the number ofcycles.

The long products thus produced will act as templates for one or theother of the oligonucleotide primers during subsequent cycles and willproduce molecules of the desired sequence [S⁺ ] or [S⁻ ] These moleculeswill also function as templates for one or the other of theoligonucleotide primers, producing further [S⁺ ] and [S⁻ ], and thus achain reaction can be sustained which will result in the accumulation of[S] at an exponential rate relative to the number of cycles.

By-products formed by oligonucleotide hybridizations other than thoseintended are not self-catalytic and thus accumulate at a linear rate.

The specific sequence to be amplified, [S], can be depicteddiagrammatically as: ##STR1## The appropriate oligonucleotide primerswould be: ##STR2## so that if DNA containing [S]

    ....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz....

    ....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz....

is separated into single strands and its single strands are hybridizedto Primers 1 and 2, the following extension reactions can be catalyzedby DNA polymerase in the presence of the four deoxyribonucleosidetriphosphates: ##STR3## On denaturation of the two duplexes formed, theproducts are: ##STR4## If these four strands are allowed to rehybridizewith Primers 1 and 2 in the next cycle, the agent for polymerizationwill catalyze the following reactions: ##STR5## If the strands of theabove four duplexes are separated, the following strands are found:##STR6##

It is seen that each strand which terminates with the oligonucleotidesequence of one primer and the complementary sequence of the other isthe specific nucleic acid sequence [S] that is desired to be produced.

The steps of this process can he repeated indefinitely, being limitedonly by the amount of Primers 1 and 2, inducing agent and nucleotidespresent. The amount of original nucleic acid remains constant in theentire process, because it is not replicated. The amount of the longproducts increases linearly because they are produced only from theoriginal nucleic acid. The amount of the specific sequence increasesexponentially. Thus, the specific sequence will become the predominantspecies. This is illustrated in the following table, which indicates therelative amounts of the species theoretically present after n cycles,assuming 100% efficiency at each cycle:

    ______________________________________                                        Number of Double Strands                                                      After 0 to n Cycles                                                                                Long      Specific                                       Cycle Number                                                                             Template  Products  Sequence [S]                                   ______________________________________                                        0          1         --        --                                             1          1         1         0                                              2          1         2         1                                              3          1         3         4                                              5          1         5         26                                             10         1         10        1013                                           15         1         15        32,752                                         20         1         20        1,048,555                                      n          1         n         (2.sup.n - n - 1)                              ______________________________________                                    

When a single-stranded nucleic acid is utilized as the template, onlyone long product is formed per cycle.

As used herein, the terms "restriction endonucleases" and "restrictionenzymes" refer to bacterial enzymes which cut double-stranded DNA at ornear a specific nucleotide sequence.

The primer(s) herein may be selected by the following criteria, whichare factors to be considered, but are not exclusive or determinative.First, the primers are selected from conserved regions of the HTLVI andHTLVII genomes. The X region is the most conserved of the codingregions, and therefore, the X region was chosen for initial studies.

Secondly, the primer lacks homology with any sequences of viral genomesthat would be expected to compromise the test, those sequences forHTLVIII, for example, being published by Starcich et al., Science,227:538-540 (1985).

Third, the primer preferably lacks secondary structure formation in theamplified nucleic acid which may interfere with extension by theamplification enzyme such as E. coli DNA polymerase, preferably thatportion of the DNA polymerase referred to as the Klenow fragment. Thismay be accomplished by employing up to about 15% by weight, preferably5-10% by weight, dimethyl sulfoxide (DMSO) in the amplification mediumand/or increasing the amplification temperatures to 30°-40° C.,preferably 35°-40° C.

Fourth, the primer preferably has an approximate 50% content of guanineand cytosine, and does not contain multiple consecutive adenine andthymine residues at the 3' end of the primer which may result in lessstable hybrids. Finally, if the amplified product will be detected byuse of a restriction enzyme, the probe must have an internal(non-terminal) restriction site.

The oligonucleotide primers may be prepared using any suitable method,such as, for example, the phosphotriester and phosphodiester methodsdescribed above, or automated embodiments thereof. In one such automatedembodiment diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage et al., Tetrahedron Letters(1981), 22:1859-1862. One method for synthesizing oligonucleotides on amodified solid support is described in U.S. Pat. No. 4,458,066. It isalso possible to use a primer which has been isolated from a biologicalsource (such as a restriction endonuclease digest).

Any source of nucleic acid, in purified or nonpurified form, can beutilized as the starting nucleic acid or acids, provided it contains oris suspected of containing the specific nucleic acid sequence associatedwith HTLVI and/or HTLVII. Thus, the process may employ, for example, DNAor RNA, including messenger RNA, which DNA or RNA may be single strandedor double stranded. In the event that RNA is to be used as a template,enzymes and/or conditions optimal for reverse transcribing the templateto DNA would be utilized. In addition, a DNA-RNA hybrid which containsone strand of each may be utilized. A mixture of any of these nucleicacids may also be employed, or the nucleic acids produced from aprevious amplification reaction herein using the same or differentprimers may be so utilized. The specific nucleic acid sequence to beamplified may be only a fraction of a larger molecule or can be presentinitially as a discrete molecule, so that the specific sequenceconstitutes the entire nucleic acid. It is not necessary that thesequence to be amplified be present initially in a pure form; it may bea minor fraction of a complex mixture, such as a portion of thevirus-encoding gene contained in whole human DNA. The starting nucleicacid may contain more than one desired specific nucleic acid sequencewhich may be the same or different. Therefore, the present process isuseful not only for producing large amounts of one specific nucleic acidsequence, but also for amplifying simultaneously more than one differentspecific nucleic acid sequence located on the same or different nucleicacid molecules.

The nucleic acid(s) may be obtained from any source, for example,natural DNA or RNA from higher organisms such as animals. DNA or RNA maybe extracted from a bodily sample, such as blood, tissue material suchas chorionic villi, or amniotic cells by a variety of techniques such asthat described by Maniatis et al., Molecular Cloning: A LaboratoryManual (New York: Cold Spring Harbor Laboratory, 1982), 280-281.

If the sample is impure such as plasma, serum or blood, beforeamplification it may be treated with an amount of a reagent effective toopen the cells, fluids, tissues, viral capsids or animal cell membranesof the sample, and to expose and/or separate the strand(s) of thenucleic acid(s). This lysing and nucleic acid denaturing step to exposeand separate the strands will allow amplification to occur much morereadily. In addition, the HTLVI and HTLVII viruses need not becultivated in the sample before the sample is treated with theamplification reagents. The sample may be centrifuged to obtain buffycoats, which are then passed through a column to obtain leukocytes. Theleukocytes may then be treated to extract the nucleic acids therefromfor use as the sample to be amplified.

Any specific nucleic acid sequence can be produced by the presentprocess. It is only necessary that a sufficient number of bases at bothends of the sequence be known in sufficient detail so that twooligonucleotide primers can be prepared which will hybridize todifferent strands of the desired sequence and at relative positionsalong the sequence such that an extension product synthesized from oneprimer, when it is separated from its template (complement), can serveas a template for extension of the other primer into a nucleic acid ofdefined length. The greater the knowledge about the bases at both endsof the sequence, the greater can be the specificity of the primers forthe target nuclei acid sequence, and thus the greater the efficiency ofthe process. It will be understood that the word primer as usedhereinafter may refer to more than one primer, particularly in the casewhere there is some ambiguity in the information regarding the terminalsequence(s) of the fragment to be amplified. For instance, in the casewhere a nucleic acid sequence is inferred from protein sequenceinformation a collection of primers containing sequences representingall possible codon variations based on degeneracy of the genetic codewill be used for each strand. One primer from this collection will besubstantially conserved with the end of the desired sequence to beamplified.

The specific nucleic acid sequence is produced by using the nucleic acidcontaining that sequence as a template. If the target nucleic acidsequence of the sample contains two strands, it is necessary to separatethe strands of the nucleic acid before it can be used as the template,either as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing any suitable denaturing conditions, including physical, chemicalor enzymatic means, the word "denaturing" used herein to include allsuch means. One physical method of separating the strands of the nucleicacid involves heating the nucleic acid unit it is denatured. Typicalheat denaturation may involve temperatures ranging from about 80° to150° C. for times ranging from about 1 to 10 minutes. Strand separationmay also be induced by an enzyme from the class of enzymes known ashelicases or the enzyme RecA, which has helicase activity and in thepresence of riboATP is known to denature DNA. The reaction conditionssuitable for separating the strands of nucleic acids with helicases aredescribed by Kuhn Hoffmann-Berling, CSH-Quantitative Biology, 43:63(1978), and techniques for using RecA are reviewed in C. Radding, Ann.Rev. Genetics, 16:405-37 (1982).

If the original nucleic acid containing the sequence to be amplified issingle stranded, its complement is synthesized by adding one or twooligonucleotide primers thereto. If an appropriate single primer isadded, a primer extension product is synthesized in the presence of theprimer, an agent for polymerization, and the four nucleosidetriphosphates described below. The product will be partiallycomplementary to the single-stranded nucleic acid and will hybridizewith the nucleic acid strand to form a duplex of unequal length strandsthat may then be separated into single strands as described above toproduce two single separated complementary strands. Alternatively, twoappropriate primers may be added to the single-stranded nucleic acid andthe reaction carried out.

If the original nucleic acid constitutes the sequence to be amplified,the primer extension product(s) produced will be completely orsubstantially completely complementary to the strands of the originalnucleic acid and will hybridize therewith to form a duplex of equallength strands to be separated into single-stranded molecules.

When the complementary strands of the nucleic acid or acids areseparated, whether the nucleic acid was originally double or singlestranded, the strands are ready to be used as a template for thesynthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates to occur. Generally it occurs in a buffered aqueous solution,preferably at a pH of 7-9, most preferably about 8. Preferably, a molarexcess (for genomic nucleic acid, usually about 10⁸ : 1 primer:template)of the two oligonucleotide primers is added to the buffer containing theseparated template strands. It is understood, however, that the amountof complementary strand may not be known if the process herein is usedfor diagnostic applications, so that the amount of primer relative tothe amount of complementary strand cannot be determined with certainty.As a practical matter, however, the amount of primer added willgenerally be in molar excess over the amount of complementary strand(template) when the sequence to be amplified is contained in a mixtureof complicated long-chain nucleic acid strands. A large molar excess ispreferred to improve the efficiency of the process.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are alsoadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. for from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein "agent for polymerization"),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis reaction may occur at fromroom temperature up to a temperature above which the agent forpolymerization no longer functions. Thus, for example, if DNA polymeraseis used as the agent, the temperature is generally no greater than about40° C. Most conveniently the reaction occurs at room temperature.

The agent for polymerization may be any compound or system which willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation), which will facilitate combination of the nucleosides inthe proper manner to form the primer extension products which arecomplementary to each nucleic acid strand. Generally, the synthesis willbe initiated at the 3' end of each primer and proceed in the 5'direction along the template strand, until synthesis terminates,producing molecules of different lengths There may be agents forpolymerization, however, which initiate synthesis at the 5' end andproceed in the other direction, using the same process as describedabove.

The newly synthesized strand and its complementary nucleic acid strandwill form a double-stranded molecule under the hybridizing conditionsdescribed above if the target sequence is present, and this hybrid isused in the succeeding steps of the process. In the next step, thesample treated under hybridizing conditions is subjected to denaturingconditions using any of the procedures described above to providesingle-stranded molecules if the target sequence is present.

New nucleic acid is synthesized on the single-stranded molecules.Additional agent for polymerization, nucleosides and primers may beadded if necessary for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

The steps of denaturing and extension product synthesis can be repeatedas often as needed to amplify the target nucleic acid sequence to theextent necessary for detection. As will be described in further detailbelow, the amount of the specific nucleic acid sequence produced willaccumulate in an exponential fashion.

When it is desired to produce more than one specific nucleic acidsequence from the first nucleic acid or mixture of nucleic acids, theappropriate number of different oligonucleotide primers are utilized.For example, if two different specific nucleic acid sequences are to beproduced, four primers are utilized. Two of the primers are specific forone of the specific nucleic acid sequences and the other two primers arespecific for the second specific nucleic acid sequence. In this manner,each of the two different specific sequences can be producedexponentially by the present process.

The present invention can be performed in a step-wise fashion whereafter each step new reagents are added, or simultaneously, where allreagents are added at the initial step, or partially step-wise andpartially simultaneous, where fresh reagent is added after a givennumber of steps. If a method of denaturation, such as heat, is employedwhich will inactivate the agent for polymerization, as in the case of aheat-labile enzyme, then it is necessary to replenish the agent afterevery strand separation step. The simultaneous method may be utilizedwhen an enzymatic means is used for the strand separation step. In thesimultaneous procedure, the reaction mixture may contain, in addition tothe nucleic acid strand(s) containing the desired sequence, thestrand-separating enzyme (e.g., helicase), an appropriate energy sourcefor the strand-separating enzyme, such as rATP, the four nucleosidetriphosphates, the oligonucleotide primers in molar excess, and theagent for polymerization, e.g., Klenow fragment of E. coli DNApolymerase I.

If heat is used for denaturation in a simultaneous process, aheat-stable agent such as a thermostable polymerase may be employedwhich will operate at an elevated temperature, preferably 50°-105° C.depending on the agent, at which temperature the nucleic acid willconsist of single and double strands in equilibrium. For smaller lengthsof nucleic acid, lower temperatures of about 40°-50° C. may be employed.The upper temperature will depend on the temperature at which the enzymewill degrade or the temperature above which an insufficient level ofprimer hybridization will occur. Such a heat-stable enzyme is described,e.g., by A. S. Kaledin et al., Biokhimiya, 45, 644-651 (1980). For thisconstant temperature reaction to succeed, the primers have their 3' endswithin 6-8 base pairs of each other. Each step of the process will occursequentially notwithstanding the initial presence of all the reagents.Additional materials may be added as necessary. After the appropriatelength of time has passed to produce the desired amount of the specificnucleic acid sequence, the reaction may be halted by inactivating theenzymes in any known manner or separating the components of thereaction.

The amplification may also be carried out using a temperature-cyclingreaction wherein the temperature is increased incrementally to allow forextension, annealing and denaturation using a heat-stable enzyme. Thisprocess and the enzyme and the instrument that can be used therefor aredescribed more fully in now abandoned U.S. application Ser. No. 899,513filed Aug. 22, 1986 and U.S. application Ser. No. 899,241 filed Aug. 22,1986, and copending Ser. No. 899,061 filed Aug. 22, 1986.

The process of the present invention may be conducted continuously. Inone embodiment of an automated process, the reaction may be cycledthrough a denaturing region, a reagent addition region, and a reactionregion. In another embodiment, the enzyme used for the synthesis ofprimer extension products can be immobilized in a column. The otherreaction components can be continuously circulated by a pump through thecolumn and a heating coil in series, thus the nucleic acids produced canbe repeatedly denatured without inactivating the enzyme.

The amplified product may be detected by analyzing it by Southern blotswithout using radioactive probes. In such a process, for example, asmall sample of DNA from, e.g., peripheral blood lymphocytes containinga very low level of the HTLVI and/or II sequence is amplified, andanalyzed via a Southern blotting technique. The use of non-radioactiveprobes is facilitated by the high level of the amplified signal.

Another method of detection involves detection using a labeled probecapable of hybridizing with the amplified nucleic acid sequence anddetermining if the probe has hybridized. Such probe necessarily containsa substantially conserved nucleic acid sequence from the genome of anHTLVI and/or HTLVII virus and is selected as described above for primersand amplified sequences. Preferably the probe is selected from the Xregion of the HTLVI and/or HTLVII genomes.

One such probe method involves the oligomer restriction techniquedescribed in U.S. Pat. No. 4,683,194. In this procedure, the amplifiednucleic acid is denatured and hybridized in solution to a labeledoligonucleotide probe which hybridizes specifically to the targetsequence (spans the particular conserved region contained by theprimers) and spans at least one restriction site of interest. The duplexformed between target and probe will reconstitute the restriction site,and when cleaved with restriction enzyme, such as, e.g., BglI, PvuII, orHifI, releases a labeled probe fragment which can be resolved from thefull-length probe by gel electrophoresis. The resulting gel is thenautoradiographed. Analysis of the amplified product by this method israpid, i.e., results can be obtained in a few hours. Preferably, theprobe is 30-45 bases long and is labeled. Also, preferably therestriction enzyme is BglI, PvuII, or HinfI.

Another method which may be used to analyze the amplified product is thedot blot method. In this method, the amplified samples are spotteddirectly on a membrane and hybridized with a labeled probe. The labelmay be detected by spectroscopy, photochemistry or by biochemical,immunochemical or chemical means. Examples include enzymes such asalkaline phosphatase, a radioactive label such as ³² P, a fluorescentlabel, or biotin. In one embodiment, the probe is a biotinylated probein which the biotin is attached to a spacer arm of the formula: ##STR7##where Y is O, NH or N--CHO, x is a number from 1 to 4, and y is a numberfrom 2 to 4. The spacer arm is in turn attached to a psoralen moiety ofthe formula: ##STR8## The psoralen moiety intercalates into andcrosslinks a "gapped circle" probe as described by Courage-Tebbe et al.,Biochim. Biophys. Acta, 697 (1982) 1-5, wherein the single-strandedhybridization region of the gapped circle spans the region containedbetween the primers. The details of this biotinylation and dot blotprocedure are described more fully in commonly assigned U.S. Pat. Nos.4,582,789 and 4,617,261, the disclosures of which are incorporatedherein by reference. The biotinylated probes eliminate the need forradioactive isotopes.

Alternatively, the probe may be spotted on the membrane first underprehybridization conditions if necessary and then the amplified productis added to the pre-treated membrane under hybridization conditions, "ina reverse" dot blot format.

The dot blot procedure is more time-consuming than the oligomerrestriction method described above, because the membrane must first beprehybridized and then hybridized with the probe. However, with rapidlymutating viruses, it has the advantage that sequences containing limitedbase mismatches are still detected under appropriate hybridizingconditions, whereas with the oligomer restriction method, any virusharboring a mutation which results in the abolishment of the restrictionsite will not be detected due to the variability of the virus.

The invention herein also contemplates a kit format which comprises apackaged multicontainer unit having containers of each primer and theprobe utilized. The kit may also have a container with the agent forpolymerization to synthesize the primer extension products, such asenzymes, a container with each of the four nucleoside triphosphates, anda container with means to detect the label (such as an avidin-enzymecomplex if the label is biotin). In addition, the kit may have acontainer which includes a positive control containing one or morenucleic acids with a sequence of the HTLVI and/or HTLVII viral genomeand/or a container including a negative control without such nucleicacids. Moreover, the kit may have a container for each restrictionenzyme capable of cleaving a nucleic acid containing the target sequenceat a site contained in a sequence in the probe.

The following examples illustrate various embodiments of the inventionand are not intended to be limiting in any respect. In the examples allparts and percentages are by weight if solid and by volume if liquid andall temperatures are in degrees Centigrade, unless otherwise indicated.

EXAMPLE 1

The desired sequences to be amplified were contained in eleven coded DNAsamples obtained from Dr. Bernard Poiesz of the Regional OncologyCenter, SUNY Upstate Medical Center, Syracuse, New York 13210,identified as 194BK, 342, 367, 361, 368H, 207, 307, 308B, 323, 326 and340. The primers and the probes were selected using the X region of theHTLVI virus and, with a few mismatches, the HTLVII virus, identified byShimotohno et al., Proc. Natl. Acad. Sci. USA 81:6657-6661 (1984).

The coded samples were first cultured in the presence of interleukin-2by Dr. Poeisz to test for the presence of virus. Then, the DNA wasextracted from the samples by the following procedure:

1. 1-2×10⁸ cultured cells were lysed in tubes with 20 ml of sodiumdodecyl sulfate lysis buffer (1% SDS, 150 mM NaCl, 25 mM Na₂ EDTA).

2. 400 μl of a 5 mg/μl solution of proteinase K was added per tube andincubated at 37° C. overnight.

3. The DNA was sequentially extracted with phenol, and CHCl₃ :isoamylalcohol followed by precipitation with ethanol.

4. The DNA was spooled out on a glass rod and resuspended in 1×TE buffer(10 mM Tris, 1 mM Na₂ EDTA, pH 7.5) and dialyzed exhaustively against1×TE buffer.

I. Synthesis of Primers

The following two oligodeoxyribonucleotide primers, designated SK43 andSK44, respectively, were prepared by the method described below:

    5'-CGGATACCCAGTCTACGTGT-3' (SK43)

    5'-GAGCCGATAACGCGTCCATCG-3'(SK44)

A. Automated Synthesis Procedures: The diethylphosphoramidites,synthesized according to Beaucage and Caruthers (Tetrahedron Letters(1981) 22:1859-1862) were sequentially condensed to a nucleosidederivatized controlled pore glass support using a Biosearch SAM-1. Theprocedure included detritylation with trichloroacetic acid indichloromethane, condensation using benzotriazole as activating protondonor, and capping with acetic anhydride and dimethylaminopyridine intetrahydrofuran and pyridine. Cycle time was approximately 30 minutes.Yields at each step were essentially quantitative and were determined bycollection and spectroscopic examination of the dimethoxytrityl alcoholreleased during detritylation.

B. Oligodeoxyribonucleotide Deprotection and Purification Procedures:The solid support was removed from the column and exposed to 1 mlconcentrated ammonium hydroxide at room temperature for four hours in aclosed tube. The support was then removed by filtration and the solutioncontaining the partially protected oligodeoxynucleotide was brought to55° C. for five hours. Ammonia was removed and the residue was appliedto a preparative polyacrylamide gel. Electrophoresis was carried out at30 volts/cm for 90 minutes after which the band containing the productwas identified by UV shadowing of a fluorescent plate. The band wasexcised and eluted with 1 ml distilled water overnight at 4° C. Thissolution was applied to an Altech RP18 column and eluted with a 7-13%gradient of acetonitrile in 1% ammonium acetate buffer at pH 6.0. Theelution was monitored by UV absorbance at 260 nm and the appropriatefraction collected, quantitated by UV absorbance in a fixed volume andevaporated to dryness at room temperature in a vacuum centrifuge.

C. Characterization of Oligodeoxyribonucleotides: Test aliquots of thepurified oligonucleotides were ³² P labeled with polynucleotide kinaseand γ-³² P-ATP. The labeled compounds were examined by autoradiographyof 14-20% polyacrylamide gels after electrophoresis for 45 minutes at 50volts/cm. This procedure verifies the molecular weight. Base compositionwas determined by digestion of the oligodeoxyribonucleotide tonucleosides by use of venom diesterase and bacterial alkalinephosphatase and subsequent separation and quantitation of the derivednucleosides using a reverse phase HPLC column and a 10% acetonitrile, 1%ammonium acetate mobile phase.

II. Amplification Reaction

One microgram of DNA from each of the eleven coded DNA samples from Dr.Poiesz was added to 100 μl of buffer consisting of 10 mM Tris-HCl, pH7.5, 50 mM sodium chloride and 10 mM magnesium chloride and containing100 picomoles of Primer SK43, 100 picomoles of Primer SK44, and 150nanomoles each of dATP, dCTP, dGTP and TTP.

The resulting solution was heated to 100° C. for 10 minutes and allowedto cool to room temperature for two minutes, whereupon 2 μl containingone unit of Klenow fragment of E. coli DNA polymerase was added. Thereaction was allowed to proceed for two minutes at room temperature,after which the enzyme was inactivated by heating at 95° C. for twominutes. The denaturation, primer annealing, and extension with Klenow,two minutes per step, and adding polymerase were repeated nineteentimes.

III. Synthesis and Phosphorylation of Oligodeoxyribonucleotide Probe

A labeled DNA probe, SK45, of the sequence:

    5'-*ACGCCCTACTGGCCACCTGTCCAGAGCATCAGATCACCTG-3',

where * indicates the label, was synthesized according to the proceduresdescribed in Section 1. The probe was labeled by contacting 10 pmolethereof with 4 units of T4 polynucleotide kinase (New England Biolabs)and 50 pmole γ⁻³² P-ATP (New England Nuclear, about 7200 Ci/mmole) in a40 μreaction volume containing 70 mM Tris buffer (pH 7.6), 10 mM MgCl₂,1.5 mM spermine, and 2.5 mM dithiothreitol for 90 minutes at 37° C. Thetotal volume was then adjusted to 100 μl with 25 mM EDTA and an aliquotremoved for determination of specific activity by TCA precipitation. Thelabeled probe was concentrated using Speed-vac and purified byelectrophoresis on a 18% polyacrylamide gel (19:1 acrylamide:BIS,Bio-Rad) in Tris-boric acid-EDTA (TBE) buffer (89 mM Tris, 89 mM boricacid, 2.5 mM EDTA, pH 8.3) for 500 vhr. After localization byautoradiography, the portion of the gel containing the labeled probe wasexcised, crushed and eluted into 0.2 ml TE buffer overnight at 4° C. TCAprecipitation of the reaction product indicated that the specificactivity was 2 Ci/mmole and the final concentration was 20 pmole/ml.

IV. Hybridization/Digestion of Amplified Genomic DNA with BglI

A. Detection in Solution

Ten microliters of amplified DNA (containing the preamplificationequivalent of 71 ng of genomic DNA) was dispensed into a 1.5 mlMicrofuge tube and 20 μl of TE buffer to a final volume of 30 μl. Thesample was denatured at 95° C. for 10 minutes. Ten microliters of 0.6 MNaCl containing 0.02 pmole of SK45 probe was added to the tube, mixedgently, overlayed with mineral oil, and immediately transferred to a 56°C. heat block for one hour. Ten microliters of 50 mM MgCl₂ and 1 μl ofBglI (8 units, New England Biolabs) were added and the reannealed DNAwas digested for 30 minutes at 56° C. The reaction was stopped by adding4 μl 75 mM EDTA and 6 μl tracking dye to a final volume of 60 μl.

The mineral oil was extracted with 0.2 ml chloroform, and 13 μl of thereaction mixture (˜15 ng genomic DNA) was loaded onto a 30%polyacrylamide mini-gel (19:1, Bio-Rad) in a Hoeffer SE200 apparatus.The gel was electrophoresed at approximately 300 volts for one houruntil the bromphenol blue dye front migrated to 3.0 cm off-origin. Thetop 1.5 cm of the gel was removed-and the remaining gel was exposed atleast overnight with two intensifying screens at -70° C.

B. Detection by Dot Blot

The amplified DNA was added to a buffer of NaOH and Na₂ EDTA such thatthe final concentration was 400 mM NaOH and 25 mM Na₂ EDTA and the finalvolume was 200 μl.

A Genetran ionic membrane was wet in water and placed in a Bio-Radimmunoblot vacuum apparatus. Then a vacuum was pulled, the apparatus wasequilibrated, and the DNA sample above was loaded onto the membrane. Themembrane was washed with 20×SSPE, where SSPE is a standard bufferconsisting of NaCl, sodium phosphate, EDTA, and NaOH. The membrane wasthen removed and placed in 20×SSPE with agitation for 2-5 minutes. Themembrane was then blotted dry and exposed to UV light for six minutes tocrosslink the DNA to the membrane.

The membrane was then placed in 5 ml of a prehybridization solution(consisting of 3×SSPE, 5×Denhardt's solution, 0.5% sodium dodecylsulfate (SDS), 30% formamide brought up to 10 ml with glass-distilledwater) for 30 minutes at 42° C. with agitation. Then theprehybridization solution was squeezed out and 5 ml of a hybridizationsolution (same as prehybridization solution with 0.5 pmole of SK45added) was added. Incubation was carried out for one hour at 42° C. withagitation.

After hybridization the membrane was washed twice in 2×SSPE, 0.1% SDSfor 15 minutes at room temperature with agitation. Then it was washedwith 0.2×SSPE, 0.1% SDS for 10 minutes at 50° C. with agitation. Themembrane was blotted dry and exposed to film.

V. Discussion of Results

The autoradiographs for both detection in solution and on a membraneshowed that the HTLVI and II DNA sequences were only present in samples342 ((HTLVI), 367 (HTLVI), 361 (HTLVI), 307 (HTLVI), 308B (HTLVI), 323(HTLVII) and 326 (HTLVI). All of these samples were later found to beeither HTLVI or HTLVII positive DNAs. The other four samples were asfollows: 194BK=DNA from leukemia patient (no virus isolated),207=patient from aggressive leukemia (skin PG,29 involvement),340=patient with aggressive leukemia (different from 207), and368=HTLVIII.

Therefore, the primers employed were able to amplify the DNA to allowthe probe to detect accurately the sequence. Amplification in thepresence of 10% DMSO (minimizes secondary structure formation) at 37° C.also indicated the HTLVI and II samples as the positive samples.

EXAMPLE 2 HTLVI

The above amplification/hybridization/digestion experiment of Example 1was repeated using HTLVI-specific primers that amplify the pol region3365-3483. These primers were:

    5'-CTTCACAGTCTCTACTGTGC-3' (SK54) and

    5'-CGGCAGTTCTGTGACAGGG-3' (SK55).

The probe SK56 below was employed with the restriction enzyme PvuII:

    5'-CCGCAGCTGCACTAATGATTGAACTTGAGAAGGAT-3' (SK56).

The autoradiograph showed that the HTLVI DNA sequence was only presentin the samples identified later as HTLVI positive DNAs.

HTLVII

The amplification/hybridization/digestion experiment of Example 1 wasrepeated using HTLVII-specific primers that amplify the pol region4198-4300. These primers were:

    5'-ATCTACCTCCACCATGTCCG-3' (SK58)

    5'-TCAGGGGAACAAGGGGAGCT-3' (SK59).

The probe SK60 below was employed with the restriction enzyme HinfI:

    5'-TAAGGGAGTCTGTGTATTCATTGAAGGTGGAAATTGGGTC-3' (SK60).

The autoradiograph showed that the HTLVII DNA sequence was only presentin sample 323 identified later as an HTLVII positive DNA.

Those skilled in the art should note that the disclosure herein ofparticular embodiments of the present invention is exemplary only, andthat various other alternative, adaptations, and modifications may bemade within the scope of the present invention. Accordingly, the presentinvention is not limited to the specific embodiments as illustratedherein, and is embodied in the claims appended hereto.

What is claimed is:
 1. A kit for detecting or monitoring a nucleic acidsequence which is substantially conserved among the isolates of bothHTLVI and HTLVII virus nucleic acids which nucleic acid sequence isspecific to the nucleic acids in both HTLVI and HTLVII isolates andwhich nucleic acid sequence is suspected of being contained in a sample,which kit comprises:(a) two oligonucleotide primers, wherein the primersare:

    5'-CGGATACCCAGTCTACGTGT-3' and

    5'-GAGCCGATAACGCGTCCATCG-3', and

(b) a probe, wherein the probe is

    5'-ACGCCCTACTGGCCACCTGTCCAGAGCATCAGATCACCTG-3'.


2. A kit for detecting or monitoring an HTLVI virus nucleic acidsequence, which HTLVI nucleic acid sequence is specific to the nucleicacids in HTLVI isolates, and which nucleic acid sequence is suspected ofbeing contained in a sample, which kit comprises:(a) two oligonucleotideprimers, wherein the primers are:

    5'-CTTCACAGTCTCTACTGTGC-3' and

    5'-CGGCAGTTCTGTGACAGGG-3'; and

(b) a probe, wherein the probe is

    5'-CCGCAGCTGCACTAATGATTGAACTTGAGAAGGAT-3'.


3. A kit for detecting or monitoring an HTLVII virus nucleic acidsequence, which HTLVII nucleic acid sequence is specific to the nucleicacids in HTLVII isolates, and which nucleic acid sequence is suspectedof being contained in a sample, which kit comprises:(a) twooligonucleotide primers, wherein the primers are:

    5'-ATCTACCTCCACCATGTCCG-3' and

    5'-TCAGGGGAACAAGGGGAGCT-3'; and

(b) a probe, wherein the probe is

    5'-TAAGGGAGTCTGTGTATTCATTGAAGGTGGAAATTGGGTC-3'.


4. An oligonucleotide primer complementary to HTLVI and/or HTLVIInucleic acids, that comprises a nucleic acid sequence between 15 and 21nucleotides in length selected from the group of nucleic acid sequencesconsisting of:

    5'-CGGATACCCAGTCTACGTGT;

    5'-GAGCCGATAACGCGTCCATCG;

    5'-CTTCACAGTCTCTACTGTGC;

    5'-CGGCAGTTCTGTGACAGGG;

    5'-ATCTACCTCCACCATGTCCG; and

    5'-TCAGGGGAACAAGGGGAGCT.


5. The primer of claim 4 that is complementary to HTLVI and HTLVIInucleic acids and comprises the nucleotide sequence5'-CGGATACCCAGTCTACGTGT-3'.
 6. The primer of claim 4 that iscomplementary to HTLVI and HTLVII nucleic acids and comprises thenucleotide sequence 5'-GAGCCGATAACGCGTCCATCG-3'.
 7. The primer of claim4 that is complementary to HTLVI nucleic acids and comprises thenucleotide sequence 5'-CTTCACAGTCTCTACTGTGC-3'.
 8. The primer of claim 4that is complementary to HTLVI nucleic acids and comprises thenucleotide sequence 5'-CGGCAGTTCTGTGACAGGG-3'.
 9. The primer of claim 4that is complementary to HTLVI nucleic acids and comprises thenucleotide sequence 5'-ATCTACCTCCACCATGTCCG-3'.
 10. The primer of claim4 that is complementary to HTLVI nucleic acids and comprises thenucleotide sequence 5'-TCAGGGGAACAAGGGGAGCT-3'.
 11. A DNA probe capableof hybridizing with an HTLVI and/or an HTLVII nucleic acid, thatcomprises a nucleic acid sequence between 15 and 40 nucleotides inlength selected from the group of nucleic acid sequences, or a sequencecomplementary thereto, consisting of:

    5'-ACGCCCTACTGGCCACCTGTCCAGAGCATCAGATCACCTG;

    5'-CCGCAGCTGCACTAATGATTGAACTTGAGAAGGAT; and

    5'-TAAGGGAGTCTGTGTATTCATTGAAGGTGGAAATTGGGTC.


12. The probe of claim 11 that is capable of hybridizing to HTLVI andHTLVII nucleic acids in a sample and comprises the nucleotide sequenceor a sequence complementary to5'-ACGCCCTACTGGCCACCTGTCCAGAGCATCAGATCACCTG-3'.
 13. The probe of claim11 that is capable of hybridizing to HTLVI nucleic acids in a sample andcomprises the nucleotide sequence or a sequence complementary to5'-CCGCAGCTGCACTAATGATTGAACTTGAGAAGGAT-3'.
 14. The probe of claim 11that is capable of hybridizing to HTLVI nucleic acids in a sample andcomprises the nucleotide sequence or a sequence complementary to5'-TAAGGGAGTCTGTGTATTCATTGAAGGTGGAAATTGGGTC-3'.